The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. This relocation of the H,K-ATPase occurs concomitantly with extensive remodeling of the actin cytoskeleton at the apical membrane, which is also an essential step in the activation of acid secretion. While these aspects of parietal cell activation are well defined, the molecular mechanisms that couple the PKA signaling cascade to mobilization of H,K-ATPases and cytoskeletal remodeling are not known. A coupling protein is ezrin, an 80 kDa phosphoprotein, whose phosphorylation by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. The long-term goal of our research is to delineate how ezrin orchestrates stimulus-coupled gastric acid secretion. To address this question, three Specific Aims are proposed: first, we will evaluate how phospho-ezrin interacts with IQGAP2 using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-IQGAP2 contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-IQGAP2 interactions in in vitro binding assays. The function of this interaction will then be determined by the effects of the peptides on acid secretion using permeabilized gastric glands. Second, we will determine how ezrin interacts with syntaxin 3 to facilitate the insertion of H,K-ATPase into the apical membrane upon the stimulation. These studies will be facilitated by real time microscopic analyses of parietal cell activation using fluorescence reporters. Third, we plan to define the role of ezrin-PALSl interaction in the apical membrane remodeling related to the cell activation by first pin-pointing their binding domains. The importance of such an interaction in acid secretion will then be evaluated by functional assay coupled with ultrastructural analysis. Studying the molecular and cellular mechanisms underlying parietal cell activation is of substantial significance in understanding the cellular physiology of regulated epithelial secretion in the gut, and is also expected to be of great benefit in leading to pharmacological strategies for correcting abnormal gastric acid secretion in disorders such as peptic ulcers, and gastroesophageal reflux disease.